Fig 1.
The HIV genome and the targets for transcription profiling assays.
This schematic shows the genetic organization of proviral HIV DNA and the HIV ‘transcription profiling’ assays targeting specific RNA sequence regions that provide insight into blocks to transcription. Some proposed mechanisms underlying the blocks to transcription initiation, elongation, and splicing are detailed.
Fig 2.
HIV RNA levels and HIV RNA/DNA ratios reveal blocks to elongation, distal transcription, and multiple-splicing in PBMCs and intact gut biopsies.
Read-through, total (TAR), 5’ elongated (R-U5/pre-Gag; “Long LTR”), Nef, polyadenylated (PolyA), and multiply-spliced Tat-Rev (MS Tat-Rev) HIV RNAs were measured in (A) PBMCs; and (B) intact rectal biopsies (n = 9 ART-suppressed individuals). 2C-D: Levels of HIV transcription per provirus are lower in the gut than the blood. Levels of each HIV RNA were normalized to HIV DNA from the same sample as measured by (C) ddPCR for the corresponding DNA sequence region (PolyA was normalized to the Read-through assay, which employs the same forward primer/probe), except for MS Tat-Rev where there is no DNA equivalent; or (D) ddPCR for the Long LTR assay, which is present once in each intact provirus. Bars indicate the median. Comparisons between transcripts were performed using the Wilcoxon signed-rank test.
Fig 3.
Stability of HIV transcripts ex vivo.
Peripheral CD4+ T cells were isolated from an ART-suppressed individual and treated with the RNA polymerase II (RNA Pol II) inhibitors (A) Triptolide [100 nM] or (B) Actinomycin D [5 mg/mL] to arrest de novo cellular and viral transcription. HIV transcripts (Read-through, TAR, Long LTR, Nef, PolyA, and MS Tat-Rev) were quantified using RT-ddPCR from cells harvested at various time points post-treatment. Levels of each HIV RNA were expressed as a proportion of the level at time t = 0 (shown) and the half-lives were determined using a one-phase exponential decay model. Data normalized to DNA mass are shown.
Fig 4.
Levels of HIV DNA, 2-LTR circles, and blocks to transcription differ between CD4+ T cells from the gut and blood.
(A) Levels of HIV DNA (Long LTR region) and 2-LTR circles were quantified in CD4+ T cells from the blood and rectum using ddPCR and expressed as copies per million CD4+ T cells (normalized by TERT). (B) The average levels per provirus of each transcript (ratio of each HIV RNA to the Long LTR HIV DNA) were measured in CD4+ T cells isolated from the blood and rectum (n = 7 matched individuals). (C) The ratio of TAR to Long LTR RNA was determined to compare the block to transcriptional elongation in CD4+ T cells from the blood and rectum. (D) The ratio of Read-through to Long LTR RNA was determined to assess the contribution of transcriptional interference to the block to HIV transcription initiation in rectal CD4+ T cells relative to peripheral CD4+ T cells. Bars represent medians. Comparisons between transcripts were performed using the Wilcoxon signed-rank test.
Fig 5.
Blocks to HIV transcription differ in the gut and blood.
Multiple blocks to HIV transcription occur in vivo and these differ in CD4+ T cells from the rectum and peripheral blood. Selected examples of potential factors are shown.
Table 1.
Characteristics of HIV-infected study participants.